Antisense compounds are an effective means for reducing the expression of specific gene products and may be uniquely useful in a number of therapeutic applications, for example, for the modulation of expression of proteins involved in fibrosis such as connective tissue growth factor (CTGF). (See U.S. Pat. No. 6,965,025B2 to Gaarde et al.)
Antisense compounds are oligomeric compounds that are capable of hybridizing to a target nucleic acid (e.g. a target mRNA molecule) and inhibiting expression of the target nucleic acid.
Antisense compounds, compositions and methods for modulating expression of CTGF and for treating diseases associated with expression of CTGF are disclosed in U.S. Pat. No. 6,965,025B2. However, there remains a need for additional such compounds capable of providing enhanced inhibition of CTGF expression as well as other advantageous properties.
Connective tissue growth factor (CTGF; also known as ctgrofact, fibroblast inducible secreted protein, fisp-12, NOV2, insulin-like growth factor-binding protein-related protein 2, IGFBP-rP2, IGFBP-8, HBGF-0.8, Hcs24, and ecogenin) is a member of the CCN (CTGF/CYR61/NOV) family of modular proteins, named for the first family members identified, connective tissue growth factor, cysteine-rich (CYR61), and nephroblastoma overexpressed (NOV), but the family also includes the proteins ELM-1 (expressed in low-metastatic cells), WISP-3 (Wnt-1-induced secreted protein), and COP-1 (WISP-2). CCN proteins have been found to be secreted, extracellular matrix-associated proteins that regulate cellular processes such as adhesion, migration, mitogenesis, differentiation, survival, angiogenesis, atherosclerosis, chondrogenesis, wound healing, tumorigenesis, and vascular and fibrotic diseases like scleroderma (Lau and Lam, Exp. Cell Res., 1999, 248, 44-57). The connective tissue growth factor protein was shown to stimulate DNA synthesis and promote chemotaxis of fibroblasts (Bradham et al., J. Cell Biol., 1991, 114, 1285-1294).
Connective tissue growth factor is expressed in fibroblasts during normal differentiation processes that involve extracellular matrix (ECM) production and remodeling. Connective tissue growth factor is also frequently overexpressed in fibrotic skin disorders such as systemic sclerosis, localized skin sclerosis, keloids, scar tissue, eosinophilic fasciitis, nodular fasciitis, and Dupuytren's contracture. Connective tissue growth factor mRNA or protein levels are elevated in fibrotic lesions of major organs and tissues including the liver, kidney, lung, cardiovascular system, pancreas, bowel, eye, and gingiva. In mammary, pancreatic and fibrohistiocytic tumors characterized by significant connective tissue involvement, connective tissue growth factor is overexpressed in the stromal compartment.
The Role of CTGF in Keloid Diseases
Keloid disease (KD) is a benign dermal fibro-proliferative tumor characterized by an excessive accumulation of extracellular matrix proteins, leading to an overabundance of collagen formation. Abnormal skin scarring can occur, post-injury in genetically susceptible individuals. KD can also be a familial condition, occurring more commonly in ethnic groups with darker skin. The highest incidence of keloids is found in the black population, where it has been estimated to be around 4-6% and up to 16% in random samples of black Africans. Various modes of inheritance have been proposed for KD ranging from autosomal recessive to autosomal dominant with incomplete clinical penetrance and variable expression. The majority of keloids can lead to considerable cosmetic defects, but can also grow large enough to become symptomatic, by causing deformity or limiting joint mobility.
Although low levels of CTGF are expressed in normal skin, CTGF becomes up-regulated following dermal injury, and it becomes persistently over-expressed when scarring is severe, as in keloids or systemic sclerosis. Fibroblasts cultured from both hypertrophic scars, keloids, and scleroderma lesions express increased basal CTGF (Exp. Cell Res. 2000, 259: 213-224), and cells cultured from hypertrophic scars and keloids were shown to express more CTGF basally and also elaborate more CTGF in response to stimulation with TGF-β (Plast. Reconstr. Surg. 2005, 116: 1387-90). Similarly, transcription of CTGF after serum stimulation was significantly higher in keloid versus normal fibroblasts in cell culture (Ann. Surg. 2007, 246(5):886-95).
In keloid tissue, fibroblasts expressing CTGF mRNA were found distributed throughout the lesions, especially in the peripheral areas (J. Invest. Derm. 1996, 106:729-733). CTGF mRNA expression levels have been compared in normal skin, keloid scars, hypertrophic scars, and mature scars. CTGF mRNA was strongly detected in all cases of the keloids, although not in mature scars. There was a significant difference between levels found in keloids and normal skin (J. Japan Soc. Plastic Reconstr. Surg. 2002, 22:560-565). Recent data also suggests that, relative to normal fibroblasts, keloid scar fibroblasts synthesize 100-150-fold more CTGF in response to exogenous TGF-β1 than do normal fibroblasts (Plast. Reconstr. Surg. 2005, 116:1387-1390). When compared to normal skin, increased localization of CTGF was seen in the basal layer of keloid epidermis and higher expression of CTGF was observed in keloid tissue extract (J. Cell Physiol. 2006, 208(2):336-43). Previously no data has been generated to validate the role of CTGF in keloid disease by showing that inhibition of CTGF expression inhibits keloid growth.
Currently, no effective single therapeutic regimen has been established for treatment of keloids or prevention of keloids growth after surgery. Existing therapeutic approaches include occlusive dressings, compression therapy, intra-lesional steroid injections, cryosurgery, surgical excision, laser treatment, radiation therapy, Kenalog (triamcinolone), interferon therapy, bleomycin, 5-flouracil, verapamil, imiquimod cream, and combinations thereof. Both silicone and non-silicone-based occlusive dressings have been a widely used clinical option for keloids for the last 30 years, but all of these methods result in very limited efficacy and it is widely understood that a new therapy for keloids is urgently needed.
Various forms of radiotherapy have been attempted as a mono-therapy for keloids, but remain quite controversial because of anecdotal reports of carcinogenesis after treatment. Laser therapy using argon, CO2, and pulse dye have been repeatedly attempted during the last 40 years, but none of them have proven to be efficacious. All three forms of laser therapy, according to multiple studies, have recurrence rates of upwards of 90%, showing little to no benefit. Cryotherapy has been used as a mono-therapy. However, side effects associated with this approach include pain at the therapeutic site and hypo- or hyper-pigmentation. Intra-lesional triamcinolone acetone injections, a type of corticosteroid, is frequently used as first-line therapy for the treatment of keloids, but again, actual reported clinical efficacy varies widely. In addition, the need for multiple injections, along with the side effects of injection pain, skin atrophy, telangiectasias, and altered pigmentation have caused clinicians and researchers to continue seeking other means of treatment.
Consequently, there remains a long felt need for additional methods and agents to effectively prevent the formation of keloids, hypertrophic scars, and other types of fibrotic lesions as well as to treat keloids, hypertrophic scars and fibrotic lesion so as to eliminate or reduce them and/or to prevent their reoccurrence. The clinical results described herein clearly demonstrate for the first time the ability of an antisense oligonucleotide targeting CTGF to reduce the growth and severity of keloids post surgery.
Antisense Dosing into Skin
It has also been demonstrated for the first time that antisense oligonucleotides do not diffuse laterally very far after dosing into skin (see Example 2) which could lead to irregular effects of this class of drug on a linear incision/healing scar or keloid if dosing was conducted as a single bolus type of administration, resulting in variable concentrations of antisense along the length of the developing scar. To overcome this drawback, a method for delivering antisense oligonucleotides by an intradermal threading technique has been developed. This technique effectively delivers a constant amount of antisense drug along the full length of the scar, and results in effective and consistent scar reduction along the full length of the scar or keloid.
Intradermal threading consists of introducing a needle into the dermis at an angle as parallel to the skin as possible, and threading the needle into and along the dermis for a distance of typically between 1 and 5 cm. At this point, the needle is withdrawn and drug injected into the dermis along the full length of the needle tract as the needle is withdrawn, resulting in an equal amount and volume of drug being deposited along the full length of the needle tract.